In mammals, challenge by bacteria and viral pathogens results in a rapid activation of the immune system to mount an expeditious, appropriate, and adequate response. The activation of specialized receptors for pathogen-associated structures on sentinel cells is translated into the production and release of endogenous alarm mediators, termed cytokines (Martin et al., 2002, Biochemica et Biophysica Acta, 1592(3):265-280). The master cytokines interleukin-1 (IL-1) and tumor necrosis factor (TNF), as well as IL-12 and IL-18, are rapidly released by these cells of first line defense. For instance, they orchestrate the acute response required to support the local phagocytes in their effort to control pathogens before they can spread systemically.
During inflammation, which results from tissue injury, infection, or autoimmune diseases, such as rheumatoid arthritis (RA), cells release these inflammatory mediators eliciting the symptoms of inflammation (see Dunne et al., 2003, Science: Signal Transduction Knowledge Environment, 2003(171):re3). These symptoms include vascular changes, such as increased blood flow, and extravasation and activation of leukocytes. During chronic inflammation, tissue remodeling and the production of acute-phase proteins by the liver occur. Cytokines are the key orchestrators of these processes, and the cytokine interleukin-1 (IL-1) plays a central role. Upon binding its receptor on the cell surface, IL-1 stimulates the expression of a large number of proinflammatory proteins. These include enzymes, such as inducible cyclooxygenase (which leads to the production of prostaglandins, themselves important inflammatory mediators), adhesion molecules, chemokines, tissue-degrading enzymes, and acute-phase proteins, such as serum amyloid A. The molecular basis for these effects has been studied in great detail, and many of the components in the signaling pathways that culminate in changes in gene expression have been delineated.
The importance of interleukin-1 in inflammation has been demonstrated by the ability of the highly specific interleukin-1 receptor antagonist protein to relieve inflammatory conditions (for review, see Dinarello (1991), Blood 77: 1627-1652; Dinarello et al. (1993), New England J. Med. 328:106-113; Dinarello (1994), FASEB J. 8:1314-1325; Dinarello (1993), Immunol. Today 14:260-264). Many of the proinflammatory effects of interleukin-1, such as the upregulation of cell adhesion molecules on vascular endothelia, are exerted at the level of transcriptional regulation. The transcriptional activation by interleukin-1 of cell adhesion molecules and other genes involved in the inflammatory response appears to be mediated largely by NF-kappa B (Shirakawa et al. (1989), Mol. Cell Biol. 9:2424-2430; Osborn et al. (1989), Proc. Natl. Acad. Sci. USA 86:2336-2340; Krasnow et al. (1991), Cytokine 3:372-379; Collins et al. (1993), Trends Cardiovasc. Med. 3:92-97). In response to interleukin-1, the NF-kappa B inhibitory factor I kappa B is degraded and NF-kappa B is released from its inactive cytoplasmic state to localize within the nucleus where it binds DNA and activates transcription (Liou et al. (1993), Curr. Opin. Cell Biol. 5:477-487; Beg et al. (1993), Mol. Cell. Bio. 13:3301-3310).
The ability of IL-1 to modify biological responses has been demonstrated in a variety of studies. For example, the administration of interleukin-1 to rabbits (Wakabayashi et al., FASEB J (1991), 5:338; Okusawa et al., J Clin Invest (1988), 81:1162; Ohlsson et al., Nature (1990), 348:550; Aiura et al., Cytokine, 1991, 4:498) and primates (Fischer et al., Am J Physiol, 1991, 261:R442) has been shown to result in hypotension, tachycardia, lung edema, renal failure, and, eventually, death, depending on the dose. When the serum from the interleukin-1 treated animals is examined, the elevation of other cytokines is evident, mimicking the levels seen in acute pancreatitis in humans. (Guice et al., J Surg Res, 1991, 51:495-499; Heath et al., Pancreas, 1993, 66:41-45). There is a body of evidence currently available which supports the role of interleukin-1 as a mediator of the systemic response to diseases such as sepsis and pancreatitis and as an activator of the remaining members of the cytokine cascade (see, e.g., Dinarello et al., Arch Surg, 1992, 127:1350-1353).
The TIR family appears to utilize similar signaling mechanisms to activate downstream effector mechanisms. While some components of the downstream signaling machinery, like the adapter TNF receptor associated factor 6 (TRAF6), are shared by other receptors of proinflammatory cytokines, one signaling module is exclusively employed by the TIR family. This consists of myeloid differentiation protein 88 (MyD88), interleukin-1 receptor associated kinase (IRAK) family members, and Toll interacting protein (Tollip).
Upon ligand binding, TIR family members form multimeric receptor complexes and via the cytoplasmic TIR domains recruit adaptor proteins such as MyD88, TIRAP and TRIF (Vogel et al., 2003, Molecular Interventions 3:466-477. This results in the sequential activation of a conserved signaling module that includes the interleukin-1 receptor associated kinases (IRAKs) and TRAF6 and activation of nuclear factor-κB (NF-κB), p38 and c-Jun N-terminal kinase (JNK) resulting in gene transcription and induction of an inflammatory response (Akira et al., 2004, Nature Reviews Immunology, 4:499-511).
Upon activation of TIR family members by ligand, IRAK4 and IRAK1 are recruited to the receptor complex. At the receptor, IRAK1 associates with the adaptors Tollip and MyD88. IRAK4 then activates IRAK1, resulting in IRAK1 autophosphorylation (Li et al., 2002, Proceedings of the National Academy of Sciences USA, 99:5567-5572; Jiang et al., 2002, Molecular and Cellular Biology 22:7158-7167). IRAK1 hyperphosphorylation results in termination of a complex with Tollip, disassociation from the receptor-MyD88 complex and formation of a protein complex of hyperphosphorylated IRAK1 and TRAF-6, a prerequisite for TRAF-6-mediated NF-κB activation and induction of an inflammatory response (Kollewe et al., 2004, Journal of Biological Chemistry, 279:5227-5236).
The IRAK family of serine-threonine kinases has four members: IRAK1, IRAK2, IRAKM and IRAK4 (Akira et al., 2004, Nature Reviews Immunology, 4:499-511; Janssens et al., 2003, Molecular Cell, 11:293-302). Despite significant structural homology between family members, they also have distinct functions. While IRAKM has been shown to function as an induced-negative regulator of TIR signaling in some contexts (Kobayashi et al., 2002, Cell, 110:191-202), other lines of evidence support a role for IRAKM as an activator of TIR-mediated signal transduction events (Wesche et al., 1999, Journal of Biological Chemistry, 274:19403-19410), leaving open the question as to the bona fide biological role played by this member of the IRAK family. IRAK2, which like IRAKM lacks kinase activity, appears to be partially redundant for IRAK1 (Kobayashi et al., 2002, Cell 110:191-202). Both IRAK1 and IRAK4 exhibit kinase activity. Both human and mouse IRAK4-deficiency results in non-responsiveness to a broad panel of TIR family ligands (Picard et al., 2003, Science 299:2076-2079; Suzuki et al., 2002, Nature, 416:750-756). IRAK1 deficiency results in a partial defect in TIR activation, with substantial decreases in IL-1, IL-18 and LPS responsiveness (Kanakaraj et al., 1998, Journal of Experimental Medicine 187:2073-2079; Kanakaraj et al., 1998, Journal of Experimental Medicine 189:1129-1138).
Due to the need for careful regulation of pro-inflammatory TIR signal transduction, multiple mechanisms exist to prevent tissue damage that arises from sustained inflammation. Given the key role that IRAK1 plays in induction of the inflammatory response, several mechanisms exist to regulate IRAK1. IRAK1 hyper-autophosphorylation results not only in full activation of kinase activity, but also in a decrease in protein stability, providing a purported mechanism by which to regulate IRAK1 activity (Yamin et al., 1997, Journal of Biological Chemistry 272:21540-21547).
Alternative splicing is another mechanism by which a single gene can generate multiple, functionally distinct protein products. A splice variant of a key adaptor protein in the TIR pathway, MyD88, can regulate signaling by serving as a dominant negative (Janssens et al., 2003, FEBS Letters, 548:103-107). Furthermore, several IRAK1 splice variants have been described in both human and mouse species. IRAK1b, which lacks 90 bp, arises from the use of an alternative 5′-acceptor splice site in exon 12. IRAK1b lacks kinase activity, but nonetheless facilitates TIR-mediated signaling, and exhibits a prolonged half-life (Jensen et al., 2001, Journal of Biological Chemistry 276:29037-29044). IRAK1s, a splice variant in mouse, is generated by a splice acceptor site in exon 12, resulting in a frame shift and generation of a premature stop codon (Yanagisawa et al., 2003, Biochemical Journal, 370:159-166). Although IRAK1s is kinase dead, it constitutively activates the NFκB and JNK pathway.
Therefore, there exists a need to identify and characterize a biologically active molecular target that functions as a dominant negative modulator of TIR signal transduction events. Furthermore, it would be advantageous to identify such a target that would be broadly applicable to the attenuation of signaling events governed by a wide range of TIR and TLR family members. Such an identified target amenable to exploitation would be useful in developing therapies directed against the many medical conditions, disorders, and diseases associated with aberrant TIR signaling activity. Furthermore, it would be beneficial to identify such a target, the expression of which can serve as a biomarker for certain of the pathologies and disorders associated with aberrant TIR signaling.